Effects of pH on Activity and Activation of Ribulose 1,5-Bisphosphate Carboxylase at Air Level CO(2)

The effects of pH on catalysis and activation characteristics of spinach ribulose 1,5-bisphosphate (RuBP) carboxylase were examined at air level of CO₂. Catalysis at limiting CO₂ was independent of pH over the range of pH 8.2 to 8.8 However, the kinetics of activation and the apparent equilibrium between the activated and inactivated forms of the enzyme were strongly dependent upon the pH and the presence or absence of the substrate RuBP. When incubated at air level of CO₂ at pH 8.2 in the absence of RuBP, the enzyme activation state was approximately 75% of that achieved with saturating CO₂ at that pH. The extent of activation increased with pH reaching 100% at pH values of 8.6 or higher. Adding RuBP to the activation medium after equilibrium activation state had been established decreased the apparent equilibrium activation level at pH values below 8.6. This effect was reversed at pH values above 8.6. Activation of inactive enzyme by CO₂ and Mg²⁺ was inhibited dramatically at pH values below 8.6 and less so at pH values above 8.6. Studies showed that binding of RuBP to the inactive form of the enzyme was pH dependent with tighter binding occurring at lower pH values. It is suggested that the tight binding of RuBP to the inactive enzyme tends to decrease the equilibrium concentration of the activated form at pH values less than 8.6. These studies indicate that stromal pH could have a strong effect on the activation state of this enzyme in vivo, and possible feedback interactions which might adjust the apparent V_max to match the rate of RuBP regeneration are discussed.     

Varying Photoperiod, Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase and CO(2) Uptake in Thalassiosira fluviatilis (Bacillariophyceae)

The purpose of this research was to test the hypothesis that acclimation of the unicellular marine alga, Thalassiosira fluviatilis Hustedt, to short photoperiods results in decreased cellular concentrations of ribulose 1,5-bisphosphate carboxylase/oxygenase and decreased rates of light-saturated CO₂ uptake. Cells were acclimated to photoperiods of 6:18, 12:12, and 18:6 h:h light:dark, and concentrations of the large subunit of the enzyme and responses of CO₂ uptake to varying irradiance were measured. Concentrations of the large subunit, which weighed approximately 50 kilodaltons, were conserved while rates of CO₂ uptake under light saturation and limitation, and cellular contents of chlorophyll a increased as photoperiod decreased. Apparently, these cells acclimate to short photoperiods by increasing rates of CO₂ uptake under saturating irradiances by increasing in vivo activation of ribulose 1,5-bisphosphate carboxylase/oxygenase. Also, chlorophyll-specific concentrations and specific activities of the enzyme appear to be lower and higher, respectively, in diatomaceous algae than in higher plants.     

Binding of Ribulose 1,5-Bisphosphate to the Non-Catalytic Sites of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase and Its Metabolic Implications

This is the first report showing that ribulose bisphosphatecarboxylase/oxygenase has the non-catalytic sites to bind ribulosebisphosphate (RuBP). A plot of the binding number against theRuBP concentration in the equilibrium binding assay gave a bumpycurve with an intermediate plateau at 0.3 to 0.5 mM RuBP. Thebinding was saturated with 1.5 mM RuBP. The concentrations offree and binding forms of RuBP and the functioning forms ofthe enzyme in chloroplasts could be predicted using the kineticdata of the binding. (Received October 5, 1993; Accepted November 22, 1993)     

Distribution of Fallover in the Carboxylase Reaction and Fallover-Inducible Sites among Ribulose 1,5-Bisphosphate Carboxylase/Oxygenases of Photosynthetic Organisms

The biphasic reaction course, fallover, of carboxyla-tion catalysedby ribulose 1,5-bisphosphate carboxylase/ox-ygenase (RuBisCO)has been known as a characteristic of the enzyme from higherland plants. Fallover consists of hysteresis in the reactionseen during the initial several minutes and a very slow suicideinhibition by inhibitors formed from the substrate ribulose-l,5-bisphosphate(RuBP). This study examined the relationship between occurrenceof fallover and non-catalytic RuBP-binding sites, and the putativehysteresis-inducible sites (Lys-21 and Lys-30S of the largesubunit in spinach RuBisCO) amongst RuBisCOs of a wide varietyof photosynthetic organisms. Fallover could be detected by followingthe course of the carboxylase reaction at 1 mM RuBP and thenon-catalytic binding sites by alleviation of fallover at 5mM RuBP. RuBisCO from Euglena gracilis showed the same linearreaction course at both RuBP concentrations, indicating an associationbetween an absence of fallover and an absence of the non-catalyticbinding sites. This was supported by the results of an equilibriumbinding assay for this enzyme with a transition state analogue.Green macroalgae and non-green algae contained the plant-type,fallover enzyme. RuBisCOs from Conjugatae, Closterium ehrenbergii,Gona-tozygon monotaenium and Netrium digitus, showed a muchsmaller decrease in activity at 1 mM RuBP than the spinach enzymeand the reaction courses of these enzymes at 5 mM RuBP werealmost linear. RuBisCO of a primitive type Conjugatae, Mesotaeniumcaldariorum, showed the same linear course at both RuBP concentrations.Sequencing of rbcL of these organisms indicated that Lys-305was changed into arginine with Lys-21 conserved. ⁷ On leave from Research and Development Center, Unitika Ltd.,23 Kozakura, Uji, Kyoto, 611 Japan. ⁸ Present address: Department of Applied Biological Chemistry,Faculty of Agriculture, Tohoku University, Tsutsumidori-Ama-miyamachi, Sendai, 981 Japan. ⁹ Present address: National Institute for Basic Biology, Myodaiji,Okazaki, 444 Japan. ¹⁰ Present address: Department of Environmental Biology, TokyoPharmaceutical University, Hachioji, Tokyo, 192-03 Japan.     

Photosynthesis and Ribulose 1,5-Bisphosphate Carboxylase in Rice Leaves: Changes in Photosynthesis and Enzymes Involved in Carbon Assimilation from Leaf Development through Senescence

Changes in photosynthesis and the ribulose 1,5-bisphosphate (RuBP) carboxylase level were examined in the 12th leaf blades of rice (Oryza sativa L.) grown under different N levels. Photosynthesis was determined using an open infrared gas analysis system. The level of RuBP carboxylase was measured by rocket immunoelectrophoresis. These changes were followed with respect to changes in the activities of RuBP carboxylase, ribulose 5-phosphate kinase, NADP-glyceraldehyde 3-phosphate dehydrogenase, and 3-phosphoglyceric acid kinase.

RuBP carboxylase activity was highly correlated with the net rate of photosynthesis (r = 0.968). Although high correlations between the activities of other enzymes and photosynthesis were also found, the activity per leaf of RuBP carboxylase was much lower than those of other enzymes throughout the leaf life. The specific activity of RuBP carboxylase on a milligram of the enzyme protein basis remained fairly constant (1.16 ± 0.07 micromoles of CO₂ per minute per milligram at 25°C) throughout the experimental period.

Kinetic parameters related to CO₂ fixation were examined using the purified carboxylase. The K_m(CO₂) and V_max values were 12 micromolar and 1.45 micromoles of CO₂ per minute per milligram, respectively (pH 8.2 and 25°C). The in vitro specific activity calculated at the atomospheric CO₂ level from the parameters was comparable to the in situ true photosynthetic rate per milligram of the carboxylase throughout the leaf life.

The results indicated that the level of RuBP carboxylase protein can be a limiting factor in photosynthesis throughout the life span of the leaf.

     

Measurement of the Enzyme-CO(2)-Mg Form of Spinach Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase

When the amount of activation of ribulose 1,5-bisphosphate carboxylase has been measured, two forms of the enzyme, not one, are actually determined experimentally. Only the enzyme-activator CO₂-Mg²⁺ form can bind ribulose bisphosphate for reaction with substrate CO₂ or O₂. A method is presented which measures only this catalytically active form by stabilizing it with ribulose bisphosphate just before dilution and assay in Mg²⁺-free reaction medium.     

Differential Modulation of Carboxylase and Oxygenase Activities of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Released from Freshly Ruptured Spinach Chloroplasts

The carboxylase activity of ribulose 1,5-bisphosphate (RuBP)carboxylase/oxygenase released from freshly ruptured spinachchloroplasts was stimulated preferentially by Mg²⁺ while oxygenaseactivity was higher with Mn²⁺. Only Mg²⁺ could reactivate eitheractivity of desalted enzyme. The results suggest that carboxylaseand oxygenase activities of RuBP craboxylase/oxygenase can bemodulated selectively by Mg²⁺ or Mn²⁺. ¹ Present address: Department of Botany, Sri Venkateswara University,Tirupati 517 502, India. (Received March 5, 1981; Accepted June 26, 1981)     

Linearity and Functioning Forms in the Carboxylase Reaction of Spinach Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase

The reaction of spinach RuBisCO activated with CO₂ and Mg²⁺proceeded in two phases, an initial burst for a few minutesand the subsequent linear phase, in the presence of saturatingconcentrations of CO₂, ribulose 1,5-bisphosphate (RuBP), andMg²⁺. The percentage of the activity in the linear phase tothat in the initial burst was 55% with RuBisCO prepared withpolyethylene glycol, and very close to the value with the enzymereleased immediately from isolated chloro-plasts. RuBisCO preparedwith ammonium sulfate had a much larger decrease of the activityin the linear phase. The Euglena enzyme had a linear courseof reaction with time for up to 20 minutes. The K_m for CO₂ of spinach RuBisCO activated beforehand was 20µM in the initial burst, and 28 µM in the linearphase. In the carboxylase reaction initiated with inactive enzyme,the activity was initially negligible, but in 5 minutes increasedto the level observed in the linear phase of the activated enzyme.The K_m for CO₂ in the linear phase of the pre-inactivated enzymewas 70 µM. The concentration of RuBP was the immediate cause of the two-phasiccourse of the carboxylase reaction of spinach RuBisCO. The curvatureof the time course was not observed below 35 µM RuBP.The enzyme required over 88 µM RuBP for the conventionaltwo-phasic course. Further increase of the concentration ofRuBP increased the extent of the curvature, but did not startthe curvature sooner after the start of the reaction. Even ifspinach RuBisCO was in the linear phase, dilution of RuBP orits consumption by the enzymatic reaction to less than 30 µMcaused the enzyme to show the resumed biphasic reaction courseafter addition of a high concentration of RuBP. ¹This paper is the twenty-fourth in a series on PhotosyntheticCarbon Metabolism in Euglena gracilis. (Received September 19, 1988; Accepted November 25, 1988)     

Low Activation State of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Carboxysome-Defective Synechococcus Mutants

The high-CO2-requiring mutant of Synechococcus sp. PCC 7942, EK6, was obtained after extension of the C terminus of the small subunit of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco). The carboxysomes in EK6 were much larger than in the wild type, but the cellular distribution of the large and small sub-units of Rubisco was not affected. The kinetic parameters of in vitro-activated Rubisco were similar in EK6 and in the wild type. On the other hand, Rubisco appeared to be in a low state of activation in situ in EK6 cells pretreated with an air level of CO2. This was deduced from the appearance of a lag phase when carboxylation was followed with time in cells permeabilized by detergent and subsequently supplied with saturating CO2 and RuBP. Pretreatment of the cells with high CO2 virtually abolished the lag. After low-CO2 treatment, the internal RuBP pool was much higher in mutant cells than in the wild-type cells; pretreatment with high CO2 reduced the pool in mutant cells. We suggest that the high-CO2-requiring phenotype in mutants that possess aberrant carboxysomes arises from the inactivated state of Rubisco when the cells are exposed to low CO2.     

Regulation of Soybean Net Photosynthetic CO(2) Fixation by the Interaction of CO(2), O(2), and Ribulose 1,5-Diphosphate Carboxylase

Kinetic properties of soybean net photosynthetic CO₂ fixation and of the carboxylase and oxygenase activities of purified soybean (Glycine max [L.] Merr.) ribulose 1, 5-diphosphate carboxylase (EC 4.1.1.39) were examined as functions of temperature, CO₂ concentration, and O₂ concentration. With leaves, O₂ inhibition of net photosynthetic CO₂ fixation increased when the ambient leaf temperature was increased. The increased inhibition of CO₂ fixation at higher temperatures was caused by a reduced affinity of the leaf for CO₂ and an increased affinity of the leaf for O₂. With purified ribulose 1,5-diphosphate carboxylase, O₂ inhibition of CO₂ incorporation and the ratio of oxygenase activity to carboxylase activity increased with increased temperature. The increased O₂ sensitivity of the enzyme at higher temperature was caused by a reduced affinity of the enzyme for CO₂ and a slightly increased affinity of the enzyme for O₂. The similarity of the effect of temperature on the affinity of intact leaves and of ribulose 1,5-diphosphate carboxylase for CO₂ and O₂ provides further evidence that the carboxylase regulates the O₂ response of photosynthetic CO₂ fixation in soybean leaves. Based on results reported here and in the literature, a scheme outlining the stoichiometry between CO₂ and O₂ fixation in vivo is proposed.     

Acclimation of Two Tomato Species to High Atmospheric CO(2): II. Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Phosphoenolpyruvate Carboxylase

Lycopersicon esculentum Mill. cv Vedettos and Lycopersicon chmielewskii Rick, LA 1028, were exposed to two CO₂ concentrations (330 or 900 microliters per liter) for 10 weeks. The elevated CO₂ concentrations increased the initial ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity of both species for the first 5 weeks of treatment but the difference did not persist during the last 5 weeks. The activity of Mg²⁺-CO₂-activated Rubisco was higher in 900 microliters per liter for the first 2 weeks but declined sharply thereafter. After 10 weeks, leaves grown at 330 microliters per liter CO₂ had about twice the Rubisco activity compared with those grown at 900 microliters per liter CO₂. The two species showed the same trend to Rubisco declines under high CO₂ concentrations. The percent activation of Rubisco was always higher under high CO₂. The phosphoenolpyruvate carboxylase (PEPCase) activity measured in tomato leaves averaged 7.9% of the total Rubisco. PEPCase showed a similar trend with time as the initial Rubisco but with no significant difference between nonenriched and CO₂-enriched plants. Long-term exposure of tomato plants to high CO₂ was previously shown to induce a decline of photosynthetic efficiency. Based on the current study and on previous results, we propose that the decline of activated Rubisco is the main cause of the acclimation of tomato plants to high CO₂ concentrations.     

Effects of Photosynthetic Intermediates on the Activation State of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Euglena gracilis Z

The half-saturating concentration of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from Euglena gracilis Z for CO₂ in its activation by CO₂ in the presence of a saturating concentration of MgCl₂ (KJ was measured by analyzing the partial reversible inactivation of the fully activated enzyme in the medium with dilute CO₂. The K_d of the Euglena enzyme was 12.5 μm. The K,_d values were 6.3/im for the enzyme from soybean, 10.8 fiM from maize, 23.3 jiM from Scenedesmus obliquus, and 20.8 μm from Anabaena 7120. The activated state of Euglena RuBisCO was stabilized by 6-phosphogluconate, fructose 1,6-bisphosphate, and 3-phosphoglycerate in the medium containing low concentrations of CO₂. Both fructose 6-phosphate and ATP stimulated inactivation in the medium. NADPH not only stabilized the activated state of the enzyme, but also enhanced the enzyme activity over the full activity measured in the absence of NADPH. NADP⁺ did not nullify the effects of NADPH on the activation at all. The physiological significance of the effects of these photosynthetic metabolites on the activated state of Euglena RuBisCO is discussed.     

pH Dependence of the Km(CO(2)) of Ribulose 1,5-Diphosphate Carboxylase

The Km(CO(2)) values of ribulose 1,5-diphosphate carboxylase in freshly ruptured spinach (Spinacia oleracea L.) chloroplasts and in the purified form isolated from spinach leaves were found to be pH dependent. Raising the pH of the assay solution produced a substantial decrease in the Km(CO(2)) of both enzyme systems. In freshly ruptured chloroplasts at pH 7.2 the Km(CO(2)) was 25 mum, at pH 8 it decreased to 19 mum, and at pH 8.8 a further decrease to 7 mum was found. With the purified enzyme at pH 7.2 the Km(CO(2)) was 147 mum, while the corresponding Km values for pH 8 and 8.8 were 34 and 15 mum CO(2), respectively. The latter figure approximates the physiological Km(CO(2)) of 10 mum estimated for photosynthesizing leaves and intact chloroplasts. The maximum velocity for both enzyme systems at optimum substrate levels was at pH 8, but the highest calculated rate of CO(2) uptake at atmospheric CO(2) levels occurred at pH 8.8. These results support the proposal that the light-induced efflux of protons out of the chloroplast stroma may be a major factor involved with the reported in vivo light activation of ribulose 1,5-diphosphate carboxylase.     

Relationships between CO(2) Exchange Rates and Activities of Pyruvate,Pi Dikinase and Ribulose Bisphosphate Carboxylase, Chlorophyll Concentration, and Cell Volume in Maize Leaves

The relationships between CO₂-exchange rate (CER), DNA and chlorophyll (Chl) concentrations, pyruvate,Pi dikinase (PPDK) and ribulose bisphosphate carboxylase (RuBPCase) activities in ten maize (Zea mays L.) genotypes were investigated. The in vivo degrees of activation of PPDK and RuBPCase were estimated to make meaningful comparisons with CER. In leaves at a photosynthetic photon flux density (PPFD) of 720 micromoles per square meter per second, in vivo PPDK degree of activation was 80% of that of PPDK fully activated in vitro, whereas RuBPCase could not be further activated in vitro, suggesting that RuBPCase was fully activated in vivo. CER varied about 50% among the genotypes tested. Significant genetic differences were observed for the average weight of a cell (estimated by gram fresh weight per milligram DNA), but this character was not correlated with CER expressed on a fresh weight basis. CER was correlated with Chl concentration, and with estimates of the in vivo degree of activation of PPDK and RuBPCase. We concluded that in maize, CER is controlled by the metabolic components of photosynthesis rather than by membrane resistances to CO₂. If the latter factor were controlling CER, then smaller cells with higher amounts of exposed cell surface area per unit cell volume would have lower resistance to CO₂ diffusion, and therefore higher CER. When data were expressed on a DNA basis (proportional to a per cell basis), results indicated that larger cells (i.e. those with higher fresh weight per milligram DNA) have a higher content of Chl, and higher PPDK and RuBPCase activities, resulting in higher CER than in smaller cells.     

Light and CO(2) Response of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activation in Arabidopsis Leaves

The requirements for activation of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) were investigated in leaves of Arabidopsis wild-type and a mutant incapable of light activating rubisco in vivo. Upon illumination with saturating light intensities, the activation state of rubisco increased 2-fold in the wild-type and decreased in the mutant. Activation of fructose 1,6-bisphosphate phosphatase was unaffected by the mutation. Under low light, rubisco deactivated in both the wild-type and the mutant. Deactivation of rubisco in the mutant under high and low light led to the accumulation of high concentrations of ribulose 1,5-bisphosphate. Inhibiting photosynthesis with methyl viologen prevented ribulose 1,5-bisphosphate accumulation but was ineffective in restoring rubisco activation to the mutant. Net photosynthesis and the rubisco activation level were closely correlated and saturated at a lower light intensity in the mutant than in wild-type. At CO₂ concentrations between 100 and 2000 microliters per liter, the activation state was a function of the CO₂ concentration in the dark but was independent of CO₂ concentration in the light. High CO₂ concentration (1%) suppressed activation in the wild-type and deactivation in the mutant. These results support the concept that rubisco activation in vivo is not a spontaneous process but is catalyzed by a specific protein. The absence of this protein, rubisco activase, is responsible for the altered characteristics of rubisco activation in the mutant.     

Ribulose Diphosphate Carboxylase from Freshly Ruptured Spinach Chloroplasts Having an in Vivo Km[CO(2)

The properties of a form of ribulose diphosphate carboxylase having a high affinity for CO(2) have been studied. Its apparent Km(HCO(3) (-)) of 0.5 to 0.8 mm (pH 7.8) and calculated Km(CO(2)) of 11 to 18 mum are comparable to the values exhibited by intact chloroplasts during photosynthesis. This form of the enzyme was released from chloroplasts in hypotonic media and was unstable, rapidly converting to a form having a high Km(HCO(3) (-)) of 20 to 25 mm similar to that for the purified enzyme. Incubation of the enzyme with MgCl(2) and HCO(3) (-) yielded a third form with an intermediate Km(HCO(3) (-)) of 2.5 to 3.0 mm.The low Km form had sufficient activity both at air levels of CO(2) and at saturating CO(2) to account for the rates of photosynthesis by intact chloroplasts. The low Km form could be stabilized in the presence of ribose 5-phosphate, adenosine triphosphate, and MgCl(2), at low temperatures for up to 2 hours.     

Crystallization of Ribulose, 1,5-Bisphosphate Carboxylase of High Specific Activity from Anther-Derived Haploid Plants of Nicotiana tabacum

Haploid plants were generated from anther-culture of Nicotianatabacum cv. Burley Ky 14. Plants that developed from pollenwithout an intervening callus stage were grown to maturity.Ploidy of the anther-derived plants were verified by chromosomecounts in root tip squashes. Crystalline ribulose 1,5-bisphosphatecarboxylase was purified from leaves of mature haploid plantsgrown in a growth chamber. Crystals appeared to be similar tothose from the diploid parental line. The specific activityof the enzyme was higher than those reported earlier in theliterature using similar crystallization procedures in Nicotianatabacum Key words: Nicotiana, haploid, ribulose 1, 5-bisphosphate carboxylase     

Exchange Properties of the Activator CO(2) of Spinach Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

The exchange properties of the activator CO₂ of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase were characterized both in vitro with the purified enzyme, and in situ within isolated chloroplasts. Carboxyarabinitol-1,5-bisphosphate, a proposed reaction intermediate analog for the carboxylase activity of the enzyme, was used to trap the activator CO₂ on the enzyme both in vitro and in situ. Modulation of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in intact chloroplasts during a light/dark cycle was associated with a similar modulation in carboxyarabinitol-1,5-bisphosphate-trapped CO₂. The exchange kinetics of the activator CO₂ were monitored by activation of the enzyme to steady state in the presence of ¹²CO₂, followed by addition of ¹⁴CO₂ and determination of the amount of labeled CO₂ trapped on the enzyme by carboxyarabinitol-1,5-bisphosphate. Rate constants (K_obs) for exchange with both the purified enzyme (0.45 min⁻¹) and in illuminated chloroplasts (0.18 min⁻¹) were comparable to the observed rate constants for enzyme activation under the two conditions. A similar exchange of the activator CO₂ was not observed in chloroplasts in the dark. Kinetic analysis of the exchange properties of the purified enzyme were consistent with an equilibrium between active and inactive forms of the enzyme during steady state activation.     

Effect of Ear Removal on CO(2) Exchange and Activities of Ribulose Bisphosphate Carboxylase/Oxygenase and Phosphoenolpyruvate Carboxylase of Maize Hybrids and Inbred Lines

The effects of ear removal on gas exchange traits, chlorophyll, and leaf N profiles, and activities of ribulose 1,5-bisphosphate carboxylase/oxygenase and phosphoenolpyruvate carboxylase were examined using four maize hybrids (B73 × Mo17, B73 × LH38, FS854, and CB59G × LH38) and four inbred lines (B73, Mo17, LH38, and CB59G) as experimental material. A diverse genotypic response to ear removal was observed which was generally typified by (a) greatly accelerated loss of chlorophyll, leaf N, enzyme activities, and CO₂ exchange relative to controls for B73, B73 × Mo17, and B73 × LH38, (b) intermediate rate of decline for leaf constituents for FS854, LH38, and Mo17, or (c) loss of leaf constituents at similar or slower rates than for control plants for CB59G and CB59G × LH38. For all genotypes which had accelerated senescence relative to controls, loss of CO₂ exchange activity was correlated with increased internal CO₂ concentrations. Thus, it was concluded that metabolic factors and not stomatal effects were responsible for loss of CO₂ exchange activity. Loss of chlorophyll, leaf N, and enzyme activities correlated well with loss of CO₂ exchange activity only for some of the genotypes. Accelerated leaf senescence in response to ear removal for the inbred line B73 and the hybrids B73 × Mo17 and B73 × LH38, as well as the apparent delayed leaf senescence for the inbred line CB59G and the hybrid CB59G × LH38 show that the contrasting responses to ear removal, rapid versus delayed senescence, can be transmitted as dominant traits to F₁ hybrids. The intermediate response by some genotypes, and the dominance of contrasting senescence traits, suggested a relatively complex inheritance for expression of the ear removal response.     

Photosynthetic Acclimation to Elevated CO2 Occurs in Transformed Tobacco with Decreased Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Content

Inhibition of net carbon assimilation rates during growth at elevated CO2 was studied in transgenic tobacco (Nicotiana tabacum L.) plants containing zero to two copies of antisense DNA sequences to the small subunit polypeptide (rbcS) gene of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). High- and low-Rubisco tobacco plants were obtained from the selfed progeny of the original line 3 transformant (S.R. Rodermel, M.S. Abbott, L. Bogorad [1988] Cell 55: 673-681). Assimilation rates of high- and low-Rubisco tobacco plants increased 22 and 71%, respectively, when transferred from 35- to 70-Pa CO2 chamber air at 900 [mu]mol m-2 s-1 photon flux density. However, CO2-dependent increases of net carbon assimilation rates of high- and low-Rubisco plants virtually disappeared after 9 d of growth in elevated CO2 chamber air. Total above-ground dry matter production of high- and low-Rubisco plants was 28 and 53% greater, respectively, after 9 d of growth at 70 Pa compared with 35 Pa CO2. Most of this dry weight gain was due to increased specific leaf weight. Rubisco activity, Rubisco protein, and total chlorophyll were lower in both high- and low-Rubisco plants grown in enriched compared with ambient CO2 chamber air. Soluble leaf protein also decreased in response to CO2 enrichment in high- but not in low-Rubisco tobacco plants. Decreased Rubisco activities in CO2-adapted high- and low-Rubisco plants were not attributable to changes in activation state of the enzyme. Carbonic anhydrase activities and subunit levels measured with specific antibodies were similar in high- and low-Rubisco tobacco plants and were unchanged by CO2 enrichment. Collectively, these findings suggested that photosynthetic acclimation to enriched CO2 occurred in tobacco plants either with or without transgenically decreased Rubisco levels and also indicated that the down-regulation of Rubisco in CO2-adapted tobacco plants was related to decreased specific activity of this enzyme.